Elongase gene and uses thereof

Abstract

The subject invention relates to the identification of a gene involved in the elongation of polyunsaturated fatty acids containing unsaturation at the carbon 9 position (i.e., “Δ9-elongase”) and to uses thereof. In particular, Δ9-elongase may be utilized, for example, in the conversion of linoleic acid (LA, 18:2n-6) to eicosadienoic acid (EDA, 20:2n-6). The production of dihomo-γ-linolenic acid (DGLA, 20:3n-6) from eicosadienoic acid (EDA, 20:2n-6), and arachidonic acid (AA, 20:4n-6) from dihomo-γ-linolenic acid (DGLA, 20:3n-6) is then catalyzed by Δ8-desaturase and Δ5-desaturase, respectively. AA or polyunsaturated fatty acids produced therefrom may be added to pharmaceutical compositions, nutritional compositions, animal feeds, as well as other products such as cosmetics.

1 . An isolated nucleic acid molecule comprising or complementary to a nucleotide sequence encoding a polypeptide having elongase activity, wherein the amino acid sequence of said polypeptide has at least about 80% sequence identity to the amino acid sequence comprising SEQ ID NO:2. 
     
     
         2 . An isolated nucleotide sequence comprising or complementary to at least about 80% of the nucleotide sequence comprising SEQ ID NO:1. 
     
     
         3 . The isolated nucleotide sequence of  claim 2  wherein said sequence comprises SEQ ID NO:1. 
     
     
         4 . The isolated nucleotide sequence of  claim 2  or  3  wherein said sequence encodes a functionally active elongase which utilizes a polyunsaturated fatty acid as a substrate. 
     
     
         5 . The nucleotide sequence of  claim 4  wherein SEQ ID NO:1 is from  Thraustochytrium aureum  7087. 
     
     
         6 . A purified polypeptide encoded by said nucleotide sequence of  claim 1 ,  2  or  3 . 
     
     
         7 . A purified polypeptide which elongates polyunsaturated fatty acids containing unsaturation at the carbon 9 position and has at least about 80% amino acid identity to an amino acid sequence comprising SEQ ID NO:2. 
     
     
         8 . A method of producing an elongase comprising the steps of:
 a) isolating a nucleotide sequence comprising SEQ ID NO:1;   b) constructing a vector comprising: i) said isolated nucleotide sequence operably linked to ii) a regulatory sequence;   c) introducing said vector into a host cell for a time and under conditions sufficient for expression of said Δ9-elongase.   
     
     
         9 . The method of  claim 8  wherein said host cell is selected from the group consisting of a eukaryotic cell and a prokaryotic cell. 
     
     
         10 . The method of  claim 9  wherein said prokaryotic cell is selected from the group consisting of  Escherichia coli , cyanobacteria and  Bacillus subtilis.    
     
     
         11 . The method of  claim 9  wherein said eukaryotic cell is selected from the group consisting of a mammalian cell, an insect cell, a plant cell and a fungal cell. 
     
     
         12 . The method of  claim 11  wherein said fungal cell is selected from the group consisting of  Saccharomyces  spp.,  Candida  spp.,  Lipomyces  spp.,  Yarrowia  spp.,  Kluyveromyces  spp.,  Hansenula  spp.,  Aspergillus  spp.,  Penicillium  spp.,  Neurospora  spp.,  Trichoderma  spp. and  Pichia  spp. 
     
     
         13 . The method of  claim 12  wherein said fungal cell is a yeast cell selected from the group consisting of  Saccharomyces  spp.,  Candida  spp.,  Hansenula  spp. and  Pichia  spp. 
     
     
         14 . The method of  claim 13  wherein said yeast cell is  Saccharomyces cerevisiae.    
     
     
         15 . A vector comprising: a) a nucleotide sequence comprising SEQ ID NO:1 operably linked to b) a regulatory sequence. 
     
     
         16 . A host cell comprising said vector of  claim 15 . 
     
     
         17 . The host cell of  claim 16 , wherein said host cell is selected from the group consisting of a eukaryotic cell and a prokaryotic cell. 
     
     
         18 . The host cell of  claim 17 , wherein said prokaryotic cell is selected from the group consisting of  Escherichia coli , cyanobacteria and  Bacillus subtilis.    
     
     
         19 . The host cell of  claim 17 , wherein said eukaryotic cell is selected from the group consisting of a mammalian cell, an insect cell, a plant cell and a fungal cell. 
     
     
         20 . The host cell of  claim 19 , wherein said fungal cell is selected from the group consisting of  Saccharomyces  spp.,  Candida  spp.,  Lipomyces  spp.,  Yarrowia  spp.,  Kluyveromyces  spp.,  Hansenula  spp.,  Aspergillus  spp.,  Penicillium  spp.,  Neurospora  spp.,  Trichoderma  spp. and  Pichia  spp. 
     
     
         21 . The host cell of  claim 20  wherein said fungal cell is a yeast cell selected from the group consisting of  Saccharomyces  spp.,  Candida  spp.,  Hansenula  spp. and  Pichia  spp. 
     
     
         22 . The host cell of  claim 21 , wherein said host cell is  Saccharomyces cerevisiae.    
     
     
         23 . A mammalian cell comprising said vector of  claim 15 , wherein expression of said nucleotide sequence of said vector results in production of altered levels of EDA and/or ETrA, when said cell is grown in a culture media comprising at least one fatty acid selected from the group consisting of LA and ALA. 
     
     
         24 . A plant cell, plant or plant tissue comprising said vector of  claim 15 , wherein expression of said nucleotide sequence of said vector results in production of at least one polyunsaturated fatty acid by said plant cell, plant or plant tissue. 
     
     
         25 . The plant cell, plant or plant tissue of  claim 24  wherein said polyunsaturated fatty acid is selected from the group consisting of EDA and EtrA. 
     
     
         26 . One or more plant oils or fatty acids expressed by said plant cell, plant or plant tissue of  claim 24 . 
     
     
         27 . A transgenic plant comprising said vector of  claim 15 , wherein expression of said nucleotide sequence of said vector results in production of at least one polyunsaturated fatty acid in seeds of said transgenic plant. 
     
     
         28 . A method for producing a polyunsaturated fatty acid comprising the steps of:
 a) isolating a nucleotide sequence comprising SEQ ID NO:1;   b) constructing a vector comprising said isolated nucleotide sequence;   c) introducing said vector into a host cell for a time and under conditions sufficient for expression of Δ9-elongase enzyme; and   d) exposing said expressed Δ9-elongase enzyme to a substrate polyunsaturated fatty acid in order to convert said substrate to a product polyunsaturated fatty acid.   
     
     
         29 . The method according to  claim 28 , wherein said substrate polyunsaturated fatty acid is LA or ALA and said product polyunsaturated fatty acid is EDA or ETrA, respectively. 
     
     
         30 . The method according to  claim 28  further comprising the step of exposing said product polyunsaturated fatty acid to at least one desaturase in order to convert said product polyunsaturated fatty acid to another polyunsaturated fatty acid. 
     
     
         31 . The method according to  claim 30  wherein said product polyunsaturated fatty acid is EDA or ETrA and said another polyunsaturated fatty acid is DGLA or ETA, respectively. 
     
     
         32 . The method of  claim 30  further comprising the step of exposing said another polyunsaturated fatty acid to at least one additional desaturase in order to convert said another polyunsaturated fatty acid to an additional polyunsaturated fatty acid. 
     
     
         33 . The method according to  claim 32  wherein said another polyunsaturated fatty acid is DGLA or ETA and said additional polyunsaturated fatty acid is AA or EPA, respectively. 
     
     
         34 . The method of  claim 32  further comprising the step of exposing said additional polyunsaturated fatty acid to at least one additional desaturase and/or at least one additional elongase in order to convert said additional polyunsaturated fatty acid to a final polyunsaturated fatty acid. 
     
     
         35 . The method of  claim 34  wherein said additional polyunsaturated fatty acid is AA or EPA and said final polyunsaturated fatty acid is ω3-docosapentaenoic acid, ω6-docosapentaenoic acid, ADA or DHA, respectively. 
     
     
         36 . A composition comprising at least one polyunsaturated fatty acid selected from the group consisting of said product polyunsaturated fatty acid produced according to the method of  claim 28 , said another polyunsaturated fatty acid produced according to the method of  claim 30 , said additional polyunsaturated fatty acid produced according to the method of  claim 32 , and said final polyunsaturated fatty acid produced according to the method of  claim 34 . 
     
     
         37 . The composition of  claim 36  wherein said product polyunsaturated fatty acid is at least one polyunsaturated fatty acid selected from the group consisting of EDA and ETrA. 
     
     
         38 . The composition of  claim 36  wherein said another polyunsaturated fatty acid is at least one polyunsaturated fatty acid selected from the group consisting of DGLA and ETA. 
     
     
         39 . The composition of  claim 36  wherein said additional polyunsaturated fatty acid is at least one polyunsaturated fatty acid selected from the group consisting of AA and EPA. 
     
     
         40 . The composition of  claim 36  wherein said final polyunsaturated fatty acid is at least one polyunsaturated fatty acid selected from the group consisting of ω3-docosapentaenoic acid, ω6-docosapentaenoic acid, ADA and DHA. 
     
     
         41 . A method of preventing or treating a condition in a patient caused by insufficient intake of polyunsaturated fatty acids comprising administering to said patient said composition of  claim 36  in an amount sufficient to effect said prevention or treatment. 
     
     
         42 . A transgenic, non-human mammal whose genome comprises a DNA sequence encoding a Δ9-elongase, operably linked to a promoter or regulatory sequence, wherein said DNA sequence comprises SEQ ID NO:1. 
     
     
         43 . A fluid produced by said transgenic, non-human mammal of  claim 42  wherein said fluid comprises a detectable level of Δ9-elongase.